Summary

Caloric restriction (CR) can delay many age-related diseases and extend lifespan, while an increase in adiposity is associated with enhanced disease risk and accelerated aging. Among the various fat depots, the accrual of visceral fat (VF) is a common feature of aging, and has been shown to be the most detrimental on metabolic syndrome of aging in humans. We have previously demonstrated that surgical removal of VF in rats improves insulin action; thus, we set out to determine if VF removal affects longevity. We prospectively studied lifespan in three groups of rats: ad libitum-fed (AL-fed), CR (Fed 60% of AL) and a group of AL-fed rats with selective removal of VF at 5 months of age (VF-removed rats). We demonstrate that compared to AL-fed rats, VF-removed rats had a significant increase in mean (p < 0.001) and maximum lifespan (p < 0.04) and significant reduction in the incidence of severe renal disease (p < 0.01). CR rats demonstrated the greatest mean and maximum lifespan (p < 0.001) and the lowest rate of death as compared to AL-fed rats (0.13). Taken together, these observations provide the most direct evidence to date that a reduction in fat mass, specifically VF, may be one of the possible underlying mechanisms of the anti-aging effect of CR.

Caloric restriction (CR) extends lifespan in a variety of species (Weindruch, 1996). In contrast, obesity is a major risk factor for several age-related diseases and has been estimated to markedly lessen life expectancy (Fontaine et al., 2003). Visceral fat (VF) accretion occurs in obesity and with aging, and a reduction in VF is a common phenotypic change in calorie-restricted mammals (Barzilai & Gupta, 1999). VF has been shown to be the single most important determinant of metabolic syndrome (Carr et al., 2004), and its removal in rats results in improved insulin action and delays the onset of diabetes (Barzilai et al., 1999; Gabriely et al., 2002). Given the hazards associated with abdominal obesity, it seems plausible that the beneficial effects of CR on longevity may be due at least in part to an attenuation of VF (Barzilai & Gupta, 1999). Here we study the effects of VF removal on the lifespan of rats.

We prospectively studied lifespan in three groups of rats: ad libitum-fed (AL-fed), CR (Fed 60% of AL) and a group of AL-fed rats with selective removal of VF at 5 months of age (VF-removed rats). At 20 months of age, a subgroup of animals (n = 8 per group) were killed to assess body fat distribution. There was no significant difference in body weights among all three groups at the beginning of the study (8 weeks of age) nor were body weights significantly different between AL-fed and VF-removed rats throughout their lifespan (Fig. 1A). However, maximal body weight was achieved at an earlier age in AL-fed rats (69 ± 3 weeks; mean ± SD) than in VF-removed rats (79 ± 3 weeks; p < 0.001), indicating a delay in the age-related weight decline in VF-removed animals. Although VF-removed rats had significantly less VF than AL-fed animals (p < 0.05), they had similar amounts of total fat (Table 1). CR animals weighed less (Fig. 1A, p < 0.001), had less body fat (Table 1, p < 0.001) and demonstrated the greatest mean and maximum lifespan among all the experimental groups (p < 0.001).

Interestingly, a significant left shift in the survival plot was demonstrated in the VF-removed group (Fig. 1B), and statistical analysis revealed a significant increase in mean (p < 0.001) and maximum lifespan (p < 0.04) for VF-removed rats as compared to AL-fed animals (Fig. 1B). In addition, the hazard rate of death in the VF-removed group was 0.49 (51% reduction) and in the CR group was 0.13 (87% reduction) compared to the AL-fed group.

Cause of death was established only in rats whose body could be examined within 2 h of death. Post-mortem analysis, performed in 28 VF-removed and 16 AL-fed rats, showed a significant decrease in severe renal disease in VF-removed rats (48%, p < 0.01). However, rates of tumor incidence were similar between VF-removed and AL-fed rats, and there were no significant differences in the incidence of other diseases.

Our data clearly demonstrate that in mammals, VF removal and CR are associated with an increase in mean and maximum lifespan. The observation of lifespan extension with removal of VF is especially interesting as VF is a potent modulator of insulin action (Barzilai et al., 1999; Gabriely et al., 2002) and a source of harmful peptides such as resistin, Plasminogen activator inhibitor-1 (PAI-1) and angiotensinogen (Einstein et al., 2005). Indeed, we have previously shown that surgical removal of VF improved insulin sensitivity in Sprague-Dawley rats (Barzilai et al., 1999) and delayed the onset of diabetes in Zucker Diabetic Fatty rats (Gabriely et al., 2002). Furthermore, VF responded to an increase in nutrient flux with a greater expression of harmful genes such as tumor necrosis factor-alpha and PAI-1 than subcutaneous fat (Einstein et al., 2005). Therefore, in addition to improving insulin action, the benefits of VF removal may include a significant decrease in secretion of potentially harmful fat-derived peptides (Gabriely et al., 2002). While VF content was substantially lower in VF-removed rats as compared to AL-fed rats, total fat mass was not significantly different. This may be due to the fact that the amount of fat removed accounted for less than 10% of total fat, and some expansion of the subcutaneous fat depot may have occurred to compensate for lesser amounts of VF. However, we and other

The mean and maximum lifespan of CR rats was greater than that seen in VF-removed animals, suggesting that the life-prolonging benefit of CR is mediated in part by pathways other than those modulated by an attenuation of VF. By comparing median lifespans, we estimate that the contribution of CR to longevity in this model was 47 weeks, whereas VF removal was 9.5 weeks, as compared to AL-fed rats, suggesting that VF reduction offered approximately 20% of the effect of CR on longevity. Taken together, this preclinical study supports the notion that limiting VF accumulation in humans might have favorable effects on health span and, potentially, longevity.

Acknowledgments

This work was supported by grants from the National Institutes of Health (AG21654 and AG18381 to N.B.) and by the Core laboratories of the Albert Einstein Diabetes Research and Training Center (DK20541).